Absolute quantification of neuromelanin in formalin-fixed human brains using absorbance spectrophotometry

Parkinson’s disease is characterised by a visual, preferential degeneration of the pigmented neurons in the substantia nigra. These neurons are pigmented by neuromelanin which decreases in Parkinson’s disease. Not much is known about NM as it is difficult to study and quantify, primarily due to its insolubility in most solvents except alkali. Neuromelanin quantification could progress the development of biomarkers for prodromal Parkinson’s disease and provide insights into the presently unclear role of neuromelanin in Parkinson’s disease aetiology. Light microscopy with stereology can visualise pigmented neurons, but it cannot quantify neuromelanin concentrations. Absolute neuromelanin quantification using absorbance spectrophotometry is reported in the literature, but the methodology is dated and only works with fresh-frozen tissue. We have developed a quantification protocol to overcome these issues. The protocol involves breakdown of fixed tissue, dissolving the tissue neuromelanin in sodium hydroxide, and measuring the solution’s 350 nm absorbance. Up to 100 brain samples can be analysed in parallel, using as little as 2 mg of tissue per sample. We used synthetic neuromelanin to construct the calibration curve rather than substantia nigra neuromelanin. Our protocol enzymatically synthesises neuromelanin from dopamine and L-cysteine followed by high-heat ageing. This protocol enables successful lysis of the fixed substantia nigra tissue and quantification in three brains, with neuromelanin concentrations ranging from 0.23–0.55 μg/mg tissue. Quantification was highly reproducible with an interassay coefficient of variation of 6.75% (n = 5). The absorbance spectra and elemental composition of the aged synthetic neuromelanin and substantia nigra neuromelanin show excellent overlap. Our protocol can robustly and reliably measure the absolute concentration of neuromelanin in formalin-fixed substantia nigra tissue. This will enable us to study how different factors affect neuromelanin and provide the basis for further development of Parkinson’s disease biomarkers and further research into neuromelanin’s role in the brain.

could be reliably quantified in formalin-fixed samples. A methodology to synthesise neuromelanin is also described, which prevents the wastage of substantia nigra neuromelanin since it is required for the calibration curve. The methodology described here is unbiased, high-throughput, and can measure neuromelanin concentration reliably.

BEFORE START INSTRUCTIONS
This protocol will use an absorbance spectrophotometer that can measure volumes less than 200 μL.
Prepare all required buffers and solutions before starting (see materials section).
If you already have dried neuromelanin (synthetic or endogenous), skip to step 13.

Note
Add 50 mL phosphate buffer, 630 μL dopamine stock solution, 61 μL L-cysteine stock solution, and 250 μL mushroom tyrosinase stock to an Erlenmeyer flask The reagent volumes listed here yields 10 mg of neuromelanin.
The room temperature in the lab was 22°C.

2
Wrap the flask with aluminium foil and make small holes on top; this protects the reaction from light but allows exposure to air. Put the flask in an incubator for 48 hours, at 37°C, with constant shaking (120 rpm).

Expected result
The solution should appear dark at the end of the incubation.

3
Stop the reaction by decreasing the solution pH to between 3 and 4, using 33% acetic acid.

4
Keep the solution wrapped in foil, as before. Incubate for 16 hours at 95°C, in an oven.

Expected result
The solution should remain dark, but the pigment should appear more granular and settled at the bottom.

5
Transfer the solution into 50 mL polypropylene tubes. Centrifuge the solution for 15 minutes at 3800 x g. Pour off the supernatant and transfer the slurry at the bottom into a 2 mL microcentrifuge tube.

Note
The room temperature in the laboratory space was 22°C.

6
Add 1.5 mL Tris buffer and incubate for 2 hours at 37°C.

7
Centrifuge the tube for 2 minutes at 9000 x g. Pour off supernatant. 8 Add 1.5 mL sodium chloride solution and wash the pellet. Centrifuge the tube for 2 minutes at 9000 x g. Pour off supernatant.

9
Add 1.5 mL ultrapure water and wash the pellet. Centrifuge the tube for 2 minutes at 9000 x g. Pour off supernatant.
Repeat this step once more. Add 1.5 mL acetone and wash the pellet. Centrifuge the tube for 2 minutes at 9000 x g. Pour off supernatant.

Expected result
A small amount of acetone will be left behind, with the clean neuromelanin dispersed throughout.

11
Dry the neuromelanin under flowing nitrogen gas for about 24 hours.

Expected result
The acetone should be fully evaporated, and the neuromelanin should be completely dry.
The expected yield is ~80% of the starting mass of materials.

12
Store the synthetic neuromelanin at -20°C in a closed plastic tube that is wrapped with aluminium foil to prevent light exposure.

13
Note Add the frozen, dried neuromelanin to ultrapure water, adjust to pH 9.0. Sonicate in an ultrasonic ice water bath for 20 minutes. Store this solution at 4°C until use.
We recommend making a 0.5 mg/mL stock solution of neuromelanin to make the process of creating the calibration curve easier. This stock can also be used for positive controls in the quantification process.

Note
Weigh 2-20 mg of fixed brain tissue (substantia nigra or locus coeruleus) using a high-accuracy scale. Homogenise the tissue in a 1.5 mL tube, using a mini plastic pestle, until a relatively fine homogenate is formed.

Note
These instructions are for analysing a single sample of brain tissue (2-20 mg). However, many samples can be easily analysed in parallel; prepare the necessary volumes of buffers and reagents as needed (see materials section).
Negative controls Negative controls can be included, which are processed the same as the samples, except no brain tissue or synthetic neuromelanin is added at the start.

Positive controls
Positive controls can also be included, which are processed the same as the samples, except a known amount of neuromelanin is spiked at the start.

Quantify neuromelanin in a brain region
Remove 900 μL of the supernatant using a micropipette, leaving behind 100 μL in the tube. Add 900 μL phosphate buffer.
Centrifuge the tube for 10 minutes at 15,000 x g.

16.1
Create the Proteinase K digestion solution by adding Proteinase K to Tris buffer to achieve a concentration of 0.25 mg/mL Proteinase K. Keep solution on ice.

Note
Laying the tube along its length is recommended to allow adequate dispersion and digestion of the tissue.

17.1
Centrifuge the tube for 10 minutes at 12,000 x g.
Remove 900 μL of the supernatant, leaving behind 100 μL in the tube. Add 900 μL sodium chloride solution.
Centrifuge the tube for 10 minutes at 12,000 x g.

Expected result
There should be a dark pellet at the bottom of the tube. This is the neuromelanin that was in the original brain sample. The surrounding tissue has been digested by Proteinase K.
Wait for 5 minutes.
Vortex the tube for ~5 seconds. Place in a room-temperature ultrasonic water bath for 3 minutes.
Centrifuge the tube for 10 minutes at 12,000 x g.
Centrifuge the tube for 10 minutes at 12,000 x g.
Sonicate the tube in a room-temperature ultrasonic water bath for 5 minutes.

22
Incubate for 1 hour at 80°C. Shake halfway through the incubation period.

Expected result
At the end of the incubation, no neuromelanin granules should be left at the bottom of the tube. If there is, increase the incubation period until all the neuromelanin has been dissolved.

23
Centrifuge the tube for 10 minutes at 15,000 x g.
Measure the absorbance at 350 nm using an absorbance spectrophotometer; we used the NanoDrop One (Thermo Fisher Scientific). Use 2 M sodium hydroxide as the blank solution.

Note
In triplicate, add 160 μL of the 0.5 mg/mL neuromelanin stock solution, 40 μL ultrapure water, The standard curve that is suggested here is from a concentration range of 3.125 μg/mL to 100 μg/mL. It uses the synthetic neuromelanin stock solution created earlier. The neuromelanin concentrations found in the pigmented brain regions fall within the concentration range.

Neuromelanin calibration curve
Measure the absorbance at 350 nm using an absorbance spectrophotometer. Use 2 M sodium hydroxide as the blank solution.

Expected result
This data can be used to generate a calibration curve which can then be used to relate sample absorbance at 350 nm to known NM concentration.